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Dimethyl fumarate (DMF), originally proposed to treat multiple sclerosis, is considered to have a spectrum of anti-inflammatory effects that effectively control periodontitis, mainly when applied with a hydrogel delivery system. Chemokine expression by gingival fibroblasts is a significant driver of periodontitis; thus, hydrogel-based strategies to deliver DMF, which in turn dampen chemokine expression, are of potential clinical relevance. To test this approach, we have established a bioassay where chemokine expression is induced by exposing gingival fibroblast to IL1β and TNFα, or with saliva. We show herein that DMF effectively reduced the expression of CXCL8, CXCL1, CXCL2, and CCL2-and lowered the phosphorylation of ERK and JNK-without affecting cell viability. This observation was confirmed by immunoassays with CXCL8. Consistently, the forced chemokine expression in HSC2 oral squamous epithelial cells was greatly diminished by DMF. To implement our hydrogel-based delivery system, gingival fibroblasts were cocultured with gellan gum hydrogels enriched for DMF. In support of our strategy, DMF-enriched gellan gum hydrogels significantly reduced the forced chemokine expression in gingival fibroblasts. Our data suggest that DMF exerts its anti-inflammatory activity in periodontal cells when released from gellan gum hydrogels, suggesting a potential clinical relevance to control overshooting chemokine expression under chronic inflammatory conditions.

Platelet-rich fibrin (PRF) is prepared by spontaneous coagulation of fractionated blood. When squeezed between two plates, PRF is separated into solid PRF membranes and a liquid exudate, the PRF serum. The question arises regarding how much the overall activity remains in the PRF membranes and what is discarded into the PRF serum. To this end, we have exposed gingival fibroblasts to lysates prepared from PRF membranes and PRF serum, followed by bulk RNA sequencing. A total of 268 up- and 136 down-regulated genes in gingival fibroblasts exposed to PRF membrane lysates were significantly regulated under the premise of a minimum log2 with 2.5-fold change and a minus log10 significance level of two, respectively. PRF serum only caused 62 up- and 32 down-regulated genes under these conditions. Among the 46 commonly up-regulated genes were CXCL1, CXCL5, CXCL6, CXCL8, IL33, IL6, and PTGS2/COX2, stanniocalcin-1-all linked to an inflammatory response. PRF membrane lysates further increased chemokines CCL2, CCL7, CXCL2, CXCL3, and IL1R1, IL1RL1, and IL1RN, as well as the paracrine factors IL11, LIF, IGF1, BMP2, BMP6, FGF2, and CCN2/CTGF, and all hyaluronan synthases. On the other hand, PRF serum increased DKK1. The genes commonly down-regulated by PRF membrane lysates and PRF serum included interferon-induced protein with tetratricopeptide repeats (IFIT1, IFIT2, IFIT3) and odd-skipped-related transcription factors (OSR1 and OSR2), as well as FGF18 and GDF15, respectively. Taken together, PRF membrane lysates, compared to PRF serum, cause a more complex response in gingival fibroblasts, but each increased chemokine expression in gingival fibroblasts.

Solid platelet-rich fibrin (PRF), consisting of coagulated plasma from fractionated blood, has been proposed to be a suitable carrier for recombinant bone morphogenetic protein 2 (BMP2) to target mesenchymal cells during bone regeneration. However, whether solid PRF can increase the expression of BMPs in mesenchymal cells remains unknown. Proteomics analysis confirmed the presence of TGF-β1 but not BMP2 in PRF lysates. According to the existing knowledge of recombinant TGF-β1, we hypothesized that PRF can increase BMP2 expression in mesenchymal cells. To test this hypothesis, we blocked TGF-β receptor 1 kinase with SB431542 in gingival fibroblasts exposed to PRF lysates. RT-PCR and immunoassays confirmed that solid PRF lysates caused a robust SB431542-dependent increase in BMP2 expression in gingival fibroblasts. Additionally, fractions of liquid PRF, namely platelet-poor plasma (PPP) and the buffy coat (BC) layer, but not heat-denatured PPP (Alb-gel), greatly induced the expression of BMP2 in gingival fibroblasts. Even though PRF has no detectable BMPs, PRF lysates similar to recombinant TGF-β1 had the capacity to provoke canonical BMP signaling, as indicated by the nuclear translocation of Smad1/5 and the increase in its phosphorylation. Taken together, our data suggest that PRF can activate TGF-β receptor 1 kinase and consequently induce the production of BMP2 in cells of the mesenchymal lineage.

Casein and whey being food supplements have been considered to be used in oral health care products. However, the response of oral cells to micellar casein and whey powder remains unclear. Considering that milk contains the growth factor TGF-β, and lactoperoxidase was recently reported to decrease the expression of inhibitor of DNA-binding (ID) proteins, there is a rationale to assume that casein and whey can also provoke these responses in oral cells. To examine the TGF-β activity, gingival fibroblasts were exposed to reconstituted casein and whey powder from food supplement before the expression of TGF-β target genes were analyzed by reverse transcription-quantitative polymerase chain reaction. Immunoassays were performed for interleukin11 (IL11) in the cell culture supernatant and for TGF-β in the reconstituted casein and whey. We blocked TGF-β by neutralizing the antibody and the TGF-β receptor type I kinase with the inhibitor SB431542. We also showed smad3 phosphorylation and smad2/3 nuclear translocation by Western blot and immunostaining, respectively. Moreover, with reconstituted casein and whey powder, ID1 and ID3 expression analysis was evaluated in HSC2 human oral squamous carcinoma cells. We report here that casein and whey powder caused a robust increase of TGF-β target genes interleukin11 (IL11), NADPH oxidase 4 (NOX4) and proteoglycan4 (PRG4) in gingival fibroblasts that was blocked by SB431542 and the neutralizing antibody. Moreover, casein and whey powder increased the phosphorylation of smad3 and nuclear translocation of smad2/3. No changes of proliferation markers Ki67 and cyclinD1 were observed. Furthermore, reconstituted casein and whey powder decreased ID1 and ID3 expression in the HSC2 oral squamous carcinoma cells. These findings suggest that the processing of milk into casein and whey powder maintains the TGF-β activity and its capacity to regulate ID1 and ID3 genes in oral fibroblasts and oral squamous carcinoma cells, respectively. These data increase the scientific knowledge on the biological activity of casein and whey with a special emphasis on oral health.

Solid platelet-rich fibrin (PRF) is produced with centrifugation tubes designed to accelerate clotting. Thus, activated platelets may accumulate within the fibrin-rich extracellular matrix even before centrifugation is initiated. It can thus be assumed that platelets and their growth factors such as transforming growth factor-β (TGF-β) are trapped within PRF independent of their relative centrifugal force (RCF), the gravitation or g -force. To test this assumption, we prepared PRF membranes with tubes where clotting is activated by a silicone-coated interior. Tubes underwent 210 g , 650 g and 1500 g for 12 min in a horizontal centrifuge. The respective PRF membranes, either in total or separated into a platelet-poor plasma and buffy coat fraction, were subjected to repeated freeze-thawing to prepare lysates. Gingival fibroblasts were exposed to the PRF lysates to provoke the expression of TGF-β target genes. We show here that the expression of interleukin 11 (IL11) and NADPH oxidase 4 (NOX4), and Smad2/3 signaling were similarly activated by all lysates when normalized to the size of the PRF membranes. Notably, platelet-poor plasma had significantly less TGF-β activity than the buffy coat fraction at both high-speed protocols. In contrast to our original assumption, the TGF-β activity in PRF lysates produced using horizontal centrifugation follows a gradient with increasing concentration from the platelet-poor plasma towards the buffy coat layer., Competing Interests: The authors declare no competing interests.

Periodontal inflammation is associated with dying cells that potentially release metabolites helping to promote inflammatory resolution. We had shown earlier that the secretome of irradiated, dying peripheral blood mononuclear cells support in vitro angiogenesis. However, the ability of the secretome to promote inflammatory resolution remains unknown. Here, we determined the expression changes of inflammatory cytokines in murine bone marrow macrophages, RAW264.7 cells, and gingival fibroblasts exposed to the secretome obtained from γ-irradiated peripheral blood mononuclear cells in vitro by RT-PCR and immunoassays. Nuclear translocation of p65 was detected by immunofluorescence staining. Phosphorylation of p65 and degradation of IκB was determined by Western blot. The secretome of irradiated peripheral blood mononuclear cells significantly decreased the expression of IL1 and IL6 in primary macrophages and RAW264.7 cells when exposed to LPS or saliva, and of IL1, IL6, and IL8 in gingival fibroblasts when exposed to IL-1β and TNFα. These changes were associated with decreased phosphorylation and nuclear translocation of p65 but not degradation of IκB in macrophages. We also show that the lipid fraction of the secretome lowered the inflammatory response of macrophages exposed to the inflammatory cues. These results demonstrate that the secretome of irradiated peripheral blood mononuclear cells can lower an in vitro simulated inflammatory response, supporting the overall concept that the secretome of dying cells promotes inflammatory resolution.

Objective: Milk can reduce intestinal tissue damage in colitis models, and protects infants against necrotizing enterocolitis. However, whether milk can decrease inflammation related to peri-implantitis and oral mucosal dehiscence remains unclear. We therefore investigated whether or not milk and fermented by-products have any anti-inflammatory effects on the cells of the oral cavity., Material and Methods: Human gingival fibroblasts and the human oral epithelial cell line HSC2 were exposed to pasteurized human milk, pasteurized cow's milk, dairy products, and powdered milk. An inflammatory response was then provoked with IL1 and TNFα. The expression changes of IL1, IL6, and IL8 were detected by reverse transcriptase PCR and immunoassay., Results: We can report that pasteurized human milk and pasteurized cow's milk as well as yoghurt, buttermilk, sour milk, whey, and powdered milk can lower the expression of inflammatory cytokines in gingival fibroblasts being stimulated by IL1 and TNFα. A similar anti-inflammatory response to pasteurized milk and dairy products was observed with the human oral epithelial cell line HSC2., Conclusion: These data suggest that pasteurized and powdered milk, as well as fermented dairy products, display an anti-inflammatory effect on oral fibroblasts and oral epithelial cells., Clinical Relevance: Our in vitro findings provide the scientific basis to extend this research towards testing the anti-inflammatory effects of milk in a pre-clinical periodontitis and peri-implantitis model.

Objective: Milk is a rich source of transforming growth factor (TGF)-β which supports intestinal mucosal homeostasis of infants. Milk may also have beneficial effects on the integrity of the oral cavity, its being part of the gastrointestinal tract. However, it is unclear if milk and fermented milk products provoke a TGF-β response in oral cells., Material and Methods: Human gingival fibroblasts were exposed to pasteurized cow milk, yoghurt, sour milk, buttermilk and whey, followed by a reverse transcriptase polymerase chain reaction of the TGF-β target genes interleukin11 (IL11), proteoglycan4 (PRG4), and NADPH oxidase 4 (NOX4). Immunoassays were performed for IL11 and TGF-β in cell culture supernatant and milk products, respectively. Signaling was investigated with the TGF-β receptor type I kinase inhibitor SB431542., Results: We report here that pasteurized cow milk and the aqueous fractions of yoghurt, sour milk, buttermilk and whey enhanced the expression of IL11, NOX4 and PRG4 in gingival fibroblasts. Moreover, IL11 protein levels in the respective supernatant were significantly increased. Cow milk, yoghurt, sour milk and buttermilk contain approximately 1-2 ng TGF-β1, whereas active TGF-β1 is hardly detectable in whey. SB431542 reduced the response of gingival fibroblasts to pasteurized cow milk and fermented milk products based on IL11 release into the supernatant., Conclusions: These results demonstrate that gingival fibroblasts respond to pasteurized cow milk and to fermented milk products with an increased expression of TGF-β target genes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: Child dentifrices vary in their composition, with possible differential impacts on cells in the oral soft tissue. While cytotoxicity studies have been performed on adult dentifrices, no respective studies have thus far been reported on child dentifrices., Material and Methods: Seventeen commercial dentifrices for children up to 12 years of age were evaluated with respect to their in vitro cytotoxicity on gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 mouse fibroblasts. Proliferation was analyzed and live-dead staining was performed., Results: Ten child dentifrices greatly reduced cell viability with LC50 values below 5 %. Four dentifrices showed a moderate cytotoxicity with LC50 values between 5 and 20 %. Three child dentifrices showed almost no cytotoxicity with LC50 values above 95 %. The results of the assays for proliferation and live-dead staining supported these findings., Conclusions: The different composition of the child dentifrices translated into a broad spectrum of in vitro cytotoxicity on cells of the oral cavity., Clinical Relevance: The in vitro data provide the scientific foundation for further in vivo research testing the clinical relevance of the present findings., Competing Interests: Compliance with Ethical Standards This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest The authors declare that they no competing interests. Funding The work was supported by the Department of Preventive, Restorative and Pediatric Dentistry, School of Dental Medicine, University of Bern, Switzerland. Informed consent Patients were informed about the possibility of using their teeth together with adhering tissue for research purposes and consent was obtained (Kantonale Ethikkommission Bern).

Background: Whole saliva provokes a substantial pro-inflammatory response in gingival fibroblasts. This raises the question whether the salivary pellet, which is used for diagnostic purposes, also has a pro-inflammatory capacity and, if yes, what the underlying mechanisms at the molecular level are., Methods: We examined the ability of extensively washed salivary pellets to provoke the expression of chemokines in gingival fibroblasts by real-time polymerase chain reaction and immunoassays. Protein composition was determined with proteomic analysis. Endotoxins were analyzed by a Limulus assay and removed by affinity chromatography. The inhibitors TAK-242 and BAY11-7082 were used to determine the involvement of the TLR4 and NF-kB signaling, respectively. Western blot was performed to detect phosphorylated p65., Results: The experiments show that salivary pellets and the corresponding washing solution contain pro-inflammatory activity without impairing cell viability. Proteomic analysis revealed proteins with a binding capacity for lipopolysaccharides, and the Limulus assay indicated the presence of endotoxin in the salivary pellets. Blocking TLR4 with TAK-242 and depletion of endotoxins both lowered the capacity of salivary pellets to increase chemokine expression and phosphorylation of p65. BAY11-7082 suppressed chemokine expression in response to the salivary pellets. Autoclaving salivary pellets also reduced their pro-inflammatory activity., Conclusions: The data support the molecular mechanism of a TLR4-NF-kB-dependent pro-inflammatory response of the gingival fibroblasts exposed to preparations of washed salivary pellets. Together, the data indicate that the salivary pellet is rich in endotoxin but it is mainly a heat labile fraction that accounts for the chemokine expression in the bioassay.

Objectives: Artificial saliva is widely used to overcome reduced natural salivary flow. Natural saliva provokes the expression of chemokines in oral fibroblasts in vitro. However, if artificial saliva changes the expression of chemokines remains unknown., Materials and Methods: Here, we investigated the ability of Saliva Orthana®, Aldiamed®, Glandosane®, and Saliva Natura® to change the expression of chemokines in human oral fibroblasts and the human oral epithelial cell line HSC-2 by means of reverse transcription polymerase chain reaction and immunoassays. Mucins isolated from bovine submaxillary glands and recombinant human mucin 1 were included in the bioassay. Formazan formation and LIVE/DEAD® staining determined the impact of artificial saliva on cell viability. The involvement of signaling pathways was determined by pharmacologic inhibitors and Western blotting., Results: In gingival fibroblasts, Saliva Orthana®-containing mucins provoked a significantly increased expression of CXC ligand 8 (CXCL8, or interleukin 8), CXCL1, and CXCL2. Immunoassays for CXCL8 and CXCL1 confirmed the translation at the protein level. The respective dilution of artificial saliva had no impact on formazan formation and LIVE/DEAD® staining. Mucins isolated from bovine submaxillary glands also increased the panel of chemokine expression in gingival fibroblasts. BAY 11-7082, a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor, but also TAK-242, an inhibitor of toll-like receptor 4 signaling, blocked chemokine expression of Saliva Orthana® and bovine mucins. In HSC-2 cells, Glandosane® significantly increased CXCL8 expression., Conclusions: Saliva Orthana® stimulated chemokine expression in gingival fibroblasts. Mammalian mucins, but also possible contaminations with endotoxins, might contribute to the respective changes in gene expression. Epithelial cells have a differential response to artificial saliva with Glandosane® changing CXCL8 expression., Clinical Relevance: Artificial saliva can incite a cellular response, if however the changing expression of chemokines by isolated fibroblasts and epithelial cells in vitro translates into a clinical condition, is not clear.

Background: Preclinical studies support the assumption that connective tissue grafts preserve the alveolar bone from resorption; the underlying cellular mechanisms, however, remain unknown. The cellular mechanisms may be attributed to the paracrine activity of the palatal fibroblasts. It was thus reasonable to suggest that palatal connective tissue grafts reduce the formation of osteoclasts., Methods: To test this hypothesis, human palatal fibroblasts were examined for their capacity to modulate the formation of osteoclasts in murine bone marrow cultures exposed to RANKL, M-CSF and TGF-β1. Osteoclastogenesis was determined by tartrate-resistant acid phosphatase (TRAP) staining and gene expression analysis. The formation of antigen presenting cells was based on the expression of CD14 and costimmulatory molecules of antigen presenting cells. The paracrine interaction of fibroblasts and the bone marrow was modeled in vitro with inserts of cell-occlusive membranes., Results: In cocultures without cell-to-cell contact, palatal fibroblasts caused a decrease in the expression of the osteoclast marker genes in bone marrow cells; calcitonin receptors, cathepsin K, TRAP, and osteoclast-associated receptor. Also the number of TRAP positive multinucleated cells was decreased in the presence of fibroblasts. Notably, palatal fibroblasts increased the expression of CD14 and the co-stimulatory proteins CD40, CD80, and CD86 in bone marrow cells. Bone marrow cells had no considerable impact on fibroblast viability and proliferation marker genes. With regard to cell distribution, osteoclasts were most prominent in the center of the membranes, while fibroblasts accumulated immediately adjacent to the border of the insert forming a ring-like structure on the surface of the culture plate., Conclusion: The data suggest that palatal fibroblasts provide a paracrine environment that reduces osteoclastogenesis and increases markers of antigen presenting cells. Morover, the paracrine model revealed a joint activity between palatal fibroblasts and bone marrow cells visualized by the characteristic cell distribution in the two separated compartments.

Aim: Chemical decontamination increases the availability of bone grafts; however, it remains unclear whether antiseptic processing changes the biological activity of bone., Materials and Methods: Bone chips were incubated with four different antiseptic solutions including (1) povidone-iodine (0.5%), (2) chlorhexidine diguluconate (0.2%), (3) hydrogen peroxide (1%) and (4) sodium hypochlorite (0.25%). After 10 min. of incubation, changes in the capacity of the bone-conditioned medium (BCM) to modulate gene expression of gingival fibroblasts was investigated., Results: Conditioned medium obtained from freshly prepared bone chips increased the expression of TGF-β target genes interleukin 11 (IL11), proteoglycan4 (PRG4), NADPH oxidase 4 (NOX4), and decreased the expression of adrenomedullin (ADM), and pentraxin 3 (PTX3) in gingival fibroblasts. Incubation of bone chips with 0.2% chlorhexidine, followed by vigorously washing resulted in a BCM with even higher expression of IL11, PRG4 and NOX4. These findings were also detected with a decrease in cell viability and an activation of apoptosis signalling. Chlorhexidine alone, at low concentrations, increased IL11, PRG4 and NOX4 expression, independent of the TGF-β receptor I kinase activity. In contrast, 0.25% sodium hypochlorite almost entirely abolished the activity of BCM, whereas the other two antiseptic solutions, 1% hydrogen peroxide and 0.5% povidone-iodine, had relatively no impact respectively., Conclusion: These in vitro findings demonstrate that incubation of bone chips with chlorhexidine differentially affects the activity of the respective BCM compared to the other antiseptic solutions. The data further suggest that the main effects are caused by chlorhexidine remaining in the BCM after repeated washing of the bone chips., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: Autologous bone is used for augmentation in the course of oral implant placement. Bone grafts release paracrine signals that can modulate mesenchymal cell differentiation in vitro. The detailed genetic response of the bone-derived fibroblasts to these paracrine signals has remained elusive. Paracrine signals accumulate in bone-conditioned medium (BCM) prepared from porcine cortical bone chips., Materials and Methods: In this study, bone-derived fibroblasts were exposed to BCM followed by a whole genome expression profiling and downstream quantitative reverse transciptase polymerase chain reaction of the most strongly regulated genes., Results: The data show that ADM, IL11, IL33, NOX4, PRG4, and PTX3 were differentially expressed in response to BCM in bone-derived fibroblasts. The transforming growth factor beta (TGF-β) receptor 1 antagonist SB431542 blocked the effect of BCM on the expression of the gene panel, except for IL33., Conclusion: These in vitro results extend existing evidence that cortical bone chips release paracrine signals that provoke a robust genetic response in mesenchymal cells that is not exclusively mediated via the TGF-β receptor. The present data provide further insights into the process of graft consolidation.

Purpose: Whole saliva comprises components of the salivary pellicle that spontaneously forms on surfaces of implants and teeth. However, there are no studies that functionally link the salivary pellicle with a possible change in gene expression., Materials and Methods: This study examined the genetic response of oral fibroblasts exposed to the salivary pellicle and whole saliva. Oral fibroblasts were seeded onto a salivary pellicle and the respective untreated surface. Oral fibroblasts were also exposed to freshly harvested sterile-filtered whole saliva. A genome-wide microarray of oral fibroblasts was performed, followed by gene ontology screening with DAVID functional annotation clustering, KEGG pathway analysis, and the STRING functional protein association network., Results: Exposure of oral fibroblasts to saliva caused 61 genes to be differentially expressed (P < .05). Gene ontology screening assigned the respective genes into 262 biologic processes, 3 cellular components, 13 molecular functions, and 7 pathways. Most remarkable was the enrichment in the inflammatory response. None of the genes regulated by whole saliva was significantly changed when cells were placed onto a salivary pellicle., Conclusion: The salivary pellicle per se does not provoke a significant inflammatory response of oral fibroblasts in vitro, whereas sterile-filtered whole saliva does produce a strong inflammatory response.

Objective: Adhesion of osteogenic cells on titanium surfaces is a prerequisite for osseointegration. Alkali treatment can increase the hydrophilicity of titanium implant surfaces, thereby supporting the adhesion of blood components. However, it is unclear if alkali treatment also supports the adhesion of cells with a fibroblastic morphology to titanium., Materials and Methods: Here, we have used a titanium alloy (Ti-6AL-4V) processed by alkali treatment to demonstrate the impact of hydrophilicity on the adhesion of primary human gingival fibroblast and bone cells. Also included were the osteosarcoma and fibroblastoma cell lines, MG63 and L929, respectively. Cell adhesion was determined by scanning electron microscopy. We also measured viability, proliferation, and protein synthesis of the adherent cells., Results: Alkali treatment increased the adhesion of gingival fibroblasts, bone cells, and the two cell lines when seeded onto the titanium alloy surface for 1 h. At 3 h, no significant changes in cell adhesion were observed. Cells grown for 1 day on the titanium alloy surfaces processed by alkali treatment behave similarly to untreated controls with regard to viability, proliferation, and protein synthesis., Conclusion: Based on these preliminary In vitro findings, we conclude that alkali treatment can support the early adhesion of cells with fibroblastic characteristics to a titanium alloy surface., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Prolyl hydroxylases (PHD) are oxygen sensors and therefore pharmacological targets to stimulate periodontal regeneration. Here we evaluate the release profile of the PHD inhibitors dimethyloxaloylglycine and l-mimosine from bone substitutes., Materials: Dimethyloxaloylglycine and l-mimosine were lyophilised onto bone substitutes including bovine bone mineral, beta-tricalcium phosphate, and hydroxyapatite. Release kinetic was evaluated by bioassays with gingival and periodontal ligament fibroblasts. We determined the capacity of PHD inhibitors to provoke VEGF expression and to repress metabolic activity and proliferation as assessed by immunoassay, MTT conversion and (3)[H]thymidine incorporation, respectively., Results: We found that the PHD inhibitors are released from bovine bone mineral as indicated by the increase of VEGF production in gingival and periodontal ligament fibroblasts. Supernatants obtained after 1h also decreased metabolic activity and proliferation of the fibroblasts. A fibrin matrix prolonged the release of PHD inhibitors up to 192h. A similar cellular response was found when supernatants from PHD inhibitors loaded beta-tricalcium phosphate and hydroxyapatite embedded in fibrin were assessed., Conclusions: In conclusion bone substitutes can serve as carriers for PHD inhibitors that maintain their capacity to provoke a pro-angiogenic response in vitro. These findings provide the basis for preclinical studies to evaluate if this release kinetic can stimulate periodontal regeneration., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: The use of prolyl hydroxylase inhibitors such as l-mimosine (L-MIM) and dimethyloxaloylglycine (DMOG) to improve angiogenesis is a new approach for periodontal regeneration. In addition to exhibiting pro-angiogenic effects, prolyl hydroxylase inhibitors can modulate the plasminogen activator system in cells from non-oral tissues. This study assesses the effect of prolyl hydroxylase inhibitors on plasminogen activation by fibroblasts from the periodontium., Methods: Gingival and periodontal ligament fibroblasts were incubated with L-MIM and DMOG. To investigate whether prolyl hydroxylase inhibitors modulate the net plasminogen activation, kinetic assays were performed with and without interleukin (IL)-1. Moreover, plasminogen activators and the respective inhibitors were analyzed by casein zymography, immune assays, and quantitative polymerase chain reaction., Results: The kinetic assay showed that L-MIM and DMOG reduced plasminogen activation under basal and IL-1-stimulated conditions. Casein zymography revealed that the effect of L-MIM involves a decrease in urokinase-type plasminogen activator activity. In agreement with these findings, reduced levels of urokinase-type plasminogen activator and elevated levels of plasminogen activator inhibitor 1 were observed., Conclusion: L-MIM and DMOG can reduce plasminogen activation by fibroblasts from the gingiva and the periodontal ligament under basal conditions and in the presence of an inflammatory cytokine.

Objective: Chronic inflammation in periodontal disease is associated with increased plasminogen activation and elevated levels of chemokines. It is unknown whether chemokines can regulate the activation of plasminogen via modulation of plasminogen activators (PA) and the corresponding plasminogen activator inhibitors (PAI) in periodontal tissue., Design: To establish a link between chemokines and activation of plasminogen, human periodontal ligament fibroblasts (PDL) and gingival fibroblasts (GF) were incubated with IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and platelet factor-4, either alone or in the presence of the inflammatory mediators TGF-beta and IL-1. The potential of the cell lysates to activate plasminogen was based on kinetic studies with the substrate casein. Casein zymography was performed to determine the molecular sizes of the PA. Total PAI-1 in the cell-conditioned medium was quantified by immunoassay., Results: We report that the chemokines did not affect activation of plasminogen by PDL and GF. Even in the presence of TGF-beta which suppressed, and IL-1 which stimulated plasminogen activation, the chemokines had no direct effect. Inhibition of PA and plasmin, but not of matrix metalloproteinases and cysteine proteinases prevented caseinolysis. The plasminogen activation capacity of the cell lysates was represented by a single band with features of uPA. The immunoassay showed that the release of PAI-1 in PDL and GF remained unaffected by the chemokines, also when stimulated with TGF-beta., Conclusions: These results suggest that plasminogen activation by PDL and GF is not directly affected by the chemokines even in the presence of the inflammatory mediators TGF-beta and IL-1.

Gingival fibroblasts are a significant source of paracrine signals required to maintain periodontal homeostasis and to mediate pathological events linked to periodontitis and oral squamous cell carcinomas. Among the potential paracrine signals are stanniocalcin-1 (STC1), involved in oxidative stress and cellular survival; amphiregulin (AREG), a growth factor that mediates the cross-talk between immune cells and epithelial cells; chromosome 11 open reading frame 96 (C11orf96) with an unclear biologic function; and the inflammation-associated prostaglandin E synthase (PTGES). Gingival fibroblasts increasingly express these genes in response to bone allografts containing remnants of injured cells. Thus, the gene expression might be caused by the local release of damage-associated molecular patterns arising from injured cells. The aim of this study is consequently to use the established gene panel as a bioassay to measure the damage-associated activity of oral cell lysates. To this aim, we have exposed gingival fibroblasts to lysates prepared from the squamous carcinoma cell lines TR146 and HSC2, oral epithelial cells, and gingival fibroblasts. We report here that all lysates significantly increased the transcription of the entire gene panel, supported for STC1 at the protein level. Blocking TGF-β receptor 1 kinase with SB431542 only partially reduced the forced expression of STC1, AREG, and C11orf96. SB431542 even increased the PTGES expression. Together, these findings suggest that the damage signals originating from oral cells can change the paracrine activity of gingival fibroblasts. Moreover, the expression panel of genes can serve as a bioassay for testing the biocompatibility of materials for oral application. [ABSTRACT FROM AUTHOR]

Platelet-rich fibrin (PRF), the coagulated plasma of fractionated blood, is widely used to support tissue regeneration in dentistry, and the underlying cellular and molecular mechanisms are increasingly being understood. Periodontal connective tissues steadily express CXCL8, a chemokine that attracts granulocytes and lymphocytes, supporting homeostatic immunity. Even though PRF is considered to dampen inflammation, it should not be ruled out that PRF increases the expression of CXCL8 in gingival fibroblasts. To test this hypothesis, we conducted a bioassay where gingival fibroblasts were exposed to PRF lysates and the respective serum. We show here that PRF lysates and, to a lesser extent, PRF serum increased the expression of CXCL8 by the gingival fibroblasts, as confirmed by immunoassay. SB203580, the inhibitor of p38 mitogen-activated protein kinase, reduced CXCL8 expression. Consistently, PRF lysates and, to a weaker range, the PRF serum also caused phosphorylation of p38 in gingival fibroblasts. Assuming that PRF is a rich source of growth factors, the TGF-β receptor type I kinase inhibitor SB431542 decreased the PRF-induced expression and translation of CXCL8. The findings suggest that PRF lysates and the respective serum drive CXCL8 expression by activating TGF-β and p38 signaling in gingival fibroblasts. [ABSTRACT FROM AUTHOR]

Bone allografts are widely used as osteoconductive support to guide bone regrowth. Bone allografts are more than a scaffold for the immigrating cells as they maintain some bioactivity of the original bone matrix. Yet, it remains unclear how immigrating cells respond to bone allografts. To this end, we have evaluated the response of mesenchymal cells exposed to acid lysates of bone allografts (ALBA). RNAseq revealed that ALBA has a strong impact on the genetic signature of gingival fibroblasts, indicated by the increased expression of IL11, AREG, C11orf96, STC1, and GK—as confirmed by RT-PCR, and for IL11 and STC1 by immunoassays. Considering that transforming growth factor-β (TGF-β) is stored in the bone matrix and may have caused the expression changes, we performed a proteomics analysis, TGF-β immunoassay, and smad2/3 nuclear translocation. ALBA neither showed detectable TGF-β nor was the lysate able to induce smad2/3 translocation. Nevertheless, the TGF-β receptor type I kinase inhibitor SB431542 significantly decreased the expression of IL11, AREG, and C11orf96, suggesting that other agonists than TGF-β are responsible for the robust cell response. The findings suggest that IL11, AREG, and C11orf96 expression in mesenchymal cells can serve as a bioassay reflecting the bioactivity of the bone allografts. [ABSTRACT FROM AUTHOR]

Damaged cells that appear as a consequence of invasive dental procedures or in response to dental materials are supposed to release damage-associated signals. These damage-associated signals not only support tissue regeneration but might also contribute to unwanted fibrosis. The aim of this study was to identify a molecular target that reflects how fibroblasts respond to necrotic oral tissue cells. To simulate the cell damage, we prepared necrotic cell lysates by sonication of the osteocytic cell line IDG-SW3 and exposed them to gingival fibroblasts. RNAseq revealed a moderate increase in IL11 expression in the gingival fibroblasts, a pleiotropic cytokine involved in fibrosis and inflammation, and also in regeneration following trauma. Necrotic lysates of the human squamous carcinoma cell lines HSC2 and TR146, as well as of gingival fibroblasts, however, caused a robust increase in IL11 expression in the gingival fibroblasts. Consistently, immunoassay revealed significantly increased IL11 levels in the gingival fibroblasts when exposed to the respective lysates. Considering that IL11 is a TGF-β target gene, IL11 expression was partially blocked by SB431542, a TGF-β receptor type I kinase inhibitor. Moreover, lysates from the HSC2, TR146, and gingival fibroblasts caused a moderate smad2/3 nuclear translocation in the gingival fibroblasts. Taken together and based on IL11 expression, our findings show that fibroblasts are sensitive to damaged oral tissue cells. [ABSTRACT FROM AUTHOR]

Objectives: To study whether damaged epithelial cells and gingival fibroblast could affect the expression of inflammatory cytokines in healthy cells. Materials and methods: Cell suspensions were submitted to different treatments to obtain the lysates: no treatment (supernatant control), sonication, and freeze/thawing. All treatments were centrifuged, and the supernatants of the lysates were used for experimentation. Cell viability assays, RT-qPCR of IL1, IL6 and IL8, IL6 immunoassay, and immunofluorescence of NF-kB p65 were applied to verify the inflammatory crosstalk of damaged cells over healthy plated cells. Furthermore, titanium discs and collagen membranes were treated with lysates and checked for IL8 expression by RT-qPCR. Results: Lysates obtained upon sonication or freeze/thawing of oral squamous carcinoma cell lines provoked a robust increase in the expression of IL1, IL6, and IL8 by gingival fibroblasts, which was confirmed by IL6 immunoassays. Lysates obtained from the gingival fibroblasts failed to increase the expression of inflammatory cytokines in oral squamous carcinoma cells. Additionally, oral squamous carcinoma cell lysates caused the activation of the NF-kB signalling cascade in gingival fibroblasts as indicated by the phosphorylation and nuclear translocation of p65. Finally, oral squamous carcinoma cell lysates adhered to the titanium and collagen membrane surfaces and increased IL8 expression by gingival fibroblasts growing in these materials. Conclusions: Injured oral epithelial cells can release factors that incite gingival fibroblasts to become pro-inflammatory. Clinical relevance: Injuries affecting the oral mucosa generate epithelial fragments that may reach the underlying connective tissue and provoke inflammation. These injuries are routinely caused by mastication, sonication for teeth cleaning, teeth preparation, prostheses maladaptation, and implant drilling. [ABSTRACT FROM AUTHOR]

Necrotic cell damage occurs as a consequence of invasive dental procedures. Loss of membrane integrity being the hallmark of necrotic cells leads to the release of cytoplasmic and membranous components. Macrophages are predestined to respond to lysates originating from necrotic cells. Here, we implement necrotic lysates from human gingival fibroblasts, HSC2, and TR146 oral epithelial cell lines, and RAW264.7 macrophage cell lines to be tested for their potential to modulate the inflammatory response of macrophages. To this aim, necrotic cell lysates were prepared by sonication or freezing/thawing of the respective cell suspension. Necrotic cell lysates were tested for their potential to modulate the lipopolysaccharide (LPS)-induced expression of inflammatory cytokines using RAW264.7 macrophages as a bioassay. We show here that all necrotic cell lysates, independent of the origin and the preparation way, reduced the expression of IL1 and IL6 in LPS-induced RAW264.7 macrophages, most obviously shown for TR146 cells. This finding was supported in a bioassay when macrophages were exposed to poly (I:C) HMW, an agonist of TLR-3. Consistently, all necrotic lysates from gingival fibroblasts, HSC2, TR146, and RAW264.7 cells reduced the nuclear translocation of p65 in LPS-exposed macrophages. This screening approach supports the overall concept that necrotic cell lysates can modulate the inflammatory capacity of macrophages. [ABSTRACT FROM AUTHOR]

Objectives: Periodontitis is a global health burden that underlines the demand for anti-inflammatory treatment. Dalbergia melanoxylon being a rich source of flavonoids has been widely used in traditional medicine but the potential anti-inflammatory activity of its dalbergiones remains to be shown. Material and methods: We have isolated 3′-hydroxy-4,4′-dimethoxydalbergione, 4-methoxydalbergione, and 4′-hydroxy-4-methoxydalbergione from Dalbergia melanoxylon and tested their potential anti-inflammatory activity. Results: All dalbergiones are potent inhibitors of an LPS-induced inflammatory response of RAW 264.7 macrophages. This is specified by IL1β and IL6 production, and the p65 nuclear translocation. Consistently, in primary macrophages, the dalbergiones caused an M1-to-M2 polarization switch indicated by the decreased ration of IL1β and IL6 versus arginase 1 and YM1 expression. To implement oral cells, we have used gingival fibroblasts exposed to IL1β and TNFα. Consistently, all dalbergiones reduced the expression of IL6 and IL8 as well as the nuclear translocation of p65. Conclusion: These findings increase the accumulating knowledge on dalbergiones and extend it towards its capacity to lower the inflammatory response of oral cells. Clinical relevance: These findings are another piece of evidence that supports the use of herbal medicine to potentially lower inflammatory events related to dentistry. [ABSTRACT FROM AUTHOR]

Background: Milk is a rich source of natural growth factors that may support oral tissue homeostasis and wound healing. We had shown earlier that blocking TGF-β receptor type I kinase with the inhibitor SB431542 abolished the expression of IL11 and other genes in human gingival fibroblasts exposed to the aqueous fraction of milk. Our aim was to identify the entire signature of TGF-β receptor type I kinase-dependent genes regulated by the aqueous fraction of human milk. Result: RNAseq revealed 99 genes being strongly regulated by milk requiring activation of the SB431542-dependent TGF-β receptor type I kinase. Among the SB431542-dependent genes is IL11 but also cadherins, claudins, collagens, potassium channels, keratins, solute carrier family proteins, transcription factors, transmembrane proteins, tumor necrosis factor ligand superfamily members, and tetraspanin family members. When focusing on our candidate gene, we could identify D609 to suppress IL11 expression, independent of phospholipase C, sphinosine-1 phosphate synthesis, and Smad-3 phosphorylation and its nuclear translocation. In contrast, genistein and blocking phosphoinositide 3-kinases by wortmannin and LY294002 increased the milk-induced IL11 expression in gingival fibroblasts. Conclusion: Taken together, our data revealed TGF-β receptor type I kinase signaling to cause major changes of the genetic signature of gingival fibroblasts exposed to aqueous fraction of human milk. [ABSTRACT FROM AUTHOR]

Amelotin (AMTN) is a secretory calcium-binding phosphoprotein controlling the adhesion of epithelial cells to the tooth surface, forming a protective seal against the oral cavity. It can be proposed that signals released upon dentinolysis increase AMTN expression in periodontal cells, thereby helping to preserve the protective seal. Support for this assumption comes from our RNA sequencing approach showing that gingival fibroblasts exposed to acid dentin lysates (ADL) greatly increased AMTN expression. In the present study, we confirm that acid dentin lysates significantly increase AMTN in gingival fibroblasts and extend this observation towards the epithelial cell lineage by use of the HSC2 oral squamous and TR146 buccal carcinoma cell lines. AMTN immunostaining revealed an intensive signal in the nucleus of HSC2 cells exposed to acid dentin lysates. Acid dentin lysates mediate their effect via the transforming growth factor (TGF)-β type 1 receptor kinase as the antagonist SB431542 abolished the expression of AMTN in the epithelial cells and fibroblasts. Similar to what is known for fibroblasts, acid dentin lysate increased Smad-3 phosphorylation in HSC2 cells. HSC2 cells also respond to the AMTN-stimulating activity of the dentin lysate when adsorbed to gelatin. When simulating regenerative approaches, enamel matrix derivative, TGF-β1, and bone morphogenetic protein-2 also caused a robust increase in SB431542-dependent AMTN expression in HSC2. Taken together, we show here that acid dentin lysate uses the TGF-β-depended signaling pathway to support the AMTN expression in epithelial cells, possibly helping in maintaining the protective seal against the oral cavity. [ABSTRACT FROM AUTHOR]

Platelet-rich fibrin (PRF) contains a broad spectrum of bioactive molecules that can trigger several cellular responses. However, these molecules along with their upstream responses remain mostly uninvestigated. By means of proteomics we revealed that PRF lysates contain more than 650 proteins, being TGF-β one of the few growth factors found. To uncover the major target genes regulated by PRF lysates, gingival fibroblasts were exposed to lysates obtained from PRF membranes followed by a whole genome array. We identified 51 genes strongly regulated by PRF including IL11, NOX4 and PRG4 which are characteristic TGF-β target genes. RT-PCR and immunoassay analysis confirmed the TGF-β receptor I kinase-dependent increased expression of IL11, NOX4 and PRG4. The PRF-derived TGF-β activity was verified by the translocation of Smad2/3 into the nucleus along with the increased phosphorylation of Smad3. Considering that PRF is clinically used in combination with dental implants and collagen membranes, we showed here that PRF-derived TGF-β activity adsorbs to titanium implants and collagen membranes indicated by the changes in gene expression and immunoassay analysis. Our study points towards TGF-β as major target of PRF and suggest that TGF-β activity released by PRF adsorbs to titanium surface and collagen membranes [ABSTRACT FROM AUTHOR]

While oral rinses used for cosmetic purposes only do not necessarily have to be antiseptic, antimicrobial activity is required for medical indications, including oral and periodontal surgery. So the question arises—is the antimicrobial activity of oral rinses associated with any destructive changes in cell viability in vitro? To answer this question, we examined twelve oral rinses with respect to their antimicrobial and cytotoxic activity. Antimicrobial activity was screened against five bacterial strains using disc diffusion. Cytotoxicity was determined by mitochondrial reductase activity with primary gingival fibroblasts, L929 cells, and HSC-2 epithelial cells. Phase contrast microscopy and trypan blue staining were then performed to reveal cell morphology. Cells remained vital after exposure to oral rinses that were only used for cosmetic purposes. Moderate cytotoxic effects were observed for oral rinses containing 0.05% chlorhexidine, ethanol, or pegylated hydrogenated castor oil and sodium dodecyl sulfate. Other oral rinses containing 0.2% chlorhexidine and cocamidopropyl betaine exhibited strong cytotoxic and antimicrobial activity. Strong cytotoxic but moderate antimicrobial activity was observed in oral rinses containing cetylpyridinium chloride. The in vitro data show that oral rinses are heterogeneous with respect to their cytotoxic and antimicrobial effects. Based on their respective properties, oral rinses can be selected either to reduce the microbial load or for cosmetic purposes. [ABSTRACT FROM AUTHOR]

Extracorporeal irradiation sterilizes resected tumor bone used as autografts in reconstruction surgery. Therapeutic irradiation is a standard technique in head and neck cancer therapy that aims to preserve organ function. Bone irradiation has a complex, mostly inhibitory, effect on remodeling and regeneration, although the underlying mechanisms are still not fully understood. It remains unclear if extracorporeal irradiation affects the paracrine-like activity of the corresponding autografts. We recently reported that bone-conditioned medium from autogenous bone chips contains a number of factors that might affect cell activity. In the present study, we investigated the effects of extracorporeal irradiation of porcine cortical bone chips on the activity of the corresponding bone-conditioned medium. The effects of bone-conditioned medium on the expressions of transforming growth factor-beta (TGF-β) target genes in oral fibroblasts were assessed. Bone-conditioned medium from bone chips exposed to a total radiation dose up to 120 Gy did not affect expressions of TGF-β target genes, including adrenomedullin, BTB/POZ domain-containing protein 11, proteoglycan 4, NADPH oxidase 4, and interleukin 11, in oral fibroblasts. In conclusion, bone irradiation does not alter the capability of the corresponding bone-conditioned medium to provoke a robust fibroblastic cell response in vitro. (J Oral Sci 58, 325-331, 2016). [ABSTRACT FROM AUTHOR]

Background: Enamel matrix derivative (EMD) and collagen membranes (CMs) are simultaneously applied in regenerative periodontal surgery. The aim of this study is to evaluate the ability of two CMs and a collagen matrix to adsorb the activity intrinsic to EMD that provokes transforming growth factor (TGF)-β signaling in oral fibroblasts.Methods: Three commercially available collagen products were exposed to EMD or recombinant TGF-β1, followed by vigorous washing. Oral fibroblasts were either seeded directly onto collagen products or were incubated with the respective supernatant. Expression of TGF-β target genes interleukin (IL)-11 and proteoglycan 4 (PRG4) was evaluated by real time polymerase chain reaction. Proteomic analysis was used to study the fraction of EMD proteins binding to collagen.Results: EMD or TGF-β1 provoked a significant increase of IL-11 and PRG4 expression of oral fibroblasts when seeded onto collagen products and when incubated with the respective supernatant. Gene expression was blocked by the TGF-β receptor I kinase inhibitor SB431542. Amelogenin bound most abundantly to gelatin-coated culture dishes. However, incubation of palatal fibroblasts with recombinant amelogenin did not alter expression of IL-11 and PRG4.Conclusion: These in vitro findings suggest that collagen products adsorb a TGF-β receptor I kinase-dependent activity of EMD and make it available for potential target cells. [ABSTRACT FROM AUTHOR]

Background: Demineralized bone matrix (DBM) is used for the treatment of osseous defects. Conditioned medium from native bone chips can activate transforming growth factor (TGF)-p signaling in mesenchymal cells. The aim of this study is to determine whether processing of native bone into DBM affects the activity of the conditioned medium. Methods: Porcine cortical bone blocks were subjected to defatting, different concentrations of hydrochloric acid, and various temperatures. DBM was lyophilized, ground, and placed into culture medium. Human gingiva and periodontal fibroblasts were exposed to the respective conditioned medium obtained from DBM (DBCM). Changes in the expression of TGF-β target genes were determined. Results: DBCM altered the expression of TGF-(5 target genes (e.g., adrenomedullin, pentraxin 3, KN motif and ankyrin repeat domains 4, interleukin 11, fiADPH oxidase 4, and BTB [POZ] domain containing 11) by at least five-fold. The response was observed in fibroblasts from both sources. Defatting lowered the activity of DBCM. The TGF-J3 receptor type I kinase inhibitor SB431542 [4-(4-(benzo[d][l,3]dioxol-5- yl)-5-(pyridin-2-yl)-1 H-imidazol-2-yl)benzamide] but not the inhibitor of bone morphogenetic protein receptor dorsomorphin, blocked the effects of DBCM on gene expression. Moreover, conditioned medium obtained from commercial human DBM modulated the expression of TGF-β target genes. Conclusion: The findings suggest that the DBCM can activate TGF-β signaling in oral fibroblasts. [ABSTRACT FROM AUTHOR]

Genome-wide microarrays have suggested that Emdogain regulates TGF-β target genes in gingival and palatal fibroblasts. However, definitive support for this contention and the extent to which TGF-β signaling contributes to the effects of Emdogain has remained elusive. We therefore studied the role of the TGF-β receptor I (TGF-βRI) kinase to mediate the effect of Emdogain on palatal fibroblasts. Palatal fibroblasts were exposed to Emdogain with and without the inhibitor for TGF-βRI kinase, SB431542. Emdogain caused 39 coding genes to be differentially expressed in palatal fibroblasts by microarray analysis (p<0.05; >10-fold). Importantly, in the presence of the TGF-βRI kinase inhibitor SB431542, Emdogain failed to cause any significant changes in gene expression. Consistent with this mechanism, three independent TGF-βRI kinase inhibitors and a TGF-β neutralizing antibody abrogated the increased expression of IL-11, a selected Emdogain target gene. The MAPK inhibitors SB203580 and U0126 lowered the impact of Emdogain on IL-11 expression. The data support that TGF-βRI kinase activity is necessary to mediate the effects of Emdogain on gene expression in vitro. [ABSTRACT FROM AUTHOR]

Dalbergia cochinchinensis has been widely used in traditional medicine because of its flavonoids; however, the impact of the flavonoids to modulate the inflammatory response to oral cells remains to be described. For this aim, we isolated 4,7,2′-trihydroxy-4′-methoxyisoflavanol (472T4MIF) and 6,4′-dihydroxy-7-methoxyflavane (64D7MF) from the heartwood of D. cochinchinensis and confirmed the chemical structure by nuclear magnetic resonance. We show here that both flavonoids are inhibitors of an inflammatory response of murine RAW 264.7 inflammatory macrophages stimulated by LPS. This is indicated by interleukin (IL)1, IL6, and chemokine CCL2 production besides the phosphorylation of p65. Consistently, in primary murine macrophages, both flavonoids decreased the inflammatory response by lowering LPS-induced IL1 and IL6 expression. To introduce oral cells, we have used human gingival fibroblasts and provoked the inflammatory response by exposing them to IL1β and TNFα. Under these conditions, 472T4MIF, but not 64D7MF, reduced the expression of chemokines CXCL1 and CXCL2. Taken together, we identified two flavonoids that can reduce the expression of cytokines and chemokines in macrophages and fibroblastic cells. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide is a damage signal at sites of chronic inflammation. The question arises whether platelet-rich fibrin (PRF), platelet-poor plasma (PPP), and the buffy coat can neutralize hydrogen peroxide toxicity and thereby counteract local oxidative stress. In the present study, gingival fibroblasts cells were exposed to hydrogen peroxide with and without lysates obtained from PRF membranes, PPP, heated PPP (75 °C for 10 min), and the buffy coat. Cell viability was examined by trypan blue staining, live-dead staining, and formazan crystal formation. Cell apoptosis was assessed by cleaved caspase-3 Western blot analysis. Reverse transcription-quantitative polymerase chain reaction (RT-PCR) was utilized to determine the impact of PRF lysates on the expression of catalase in fibroblasts. It was reported that lysates from PRF, PPP, and the buffy coat—but not heated PPP—abolished the hydrogen peroxide-induced toxicity in gingival fibroblasts. Necrosis was confirmed by a loss of membrane integrity and apoptosis was ruled out by the lack of cleavage of caspase-3. Aminotriazole, an inhibitor of catalase, reduced the cytoprotective activity of PRF lysates yet blocking of glutathione peroxidase by mercaptosuccinate did not show the same effect. PRF lysates had no impact on the expression of catalase in gingival fibroblasts. These findings suggest that PRF, PPP, and the buffy coat can neutralize hydrogen peroxide through the release of heat-sensitive catalase. [ABSTRACT FROM AUTHOR]

Background: Demineralized bone matrix is a widely used allograft from where not only the inorganic mineral but also embedded growth factors are removed by 0.1 M hydrochloric acid. The cellular response to the growth factors released during the preparation of demineralized bone matrix has not been studied. Aim/Hypothesis: To investigate the in vitro impact of acid bone lysates (ABL) prepared from porcine cortical bone chips on oral fibroblasts. Material and Methods: Whole genome microarray was performed to screen for the ABL target genes in oral fibroblasts. Gene expression changes were confirmed by RT‐PCR and the translation into protein done by immunoassay. Immunostaining was implemented to visualize nuclear accumulation of Smad‐3. Results: Whole genome microarray together with the pharmacologic blocking of TGF‐β receptor type I kinase with SB431542 revealed that acid bony lysates activate TGF‐β signaling in oral fibroblasts. These findings were further confirmed by the SB431542‐dependent expression of interleukin 11, proteoglycan4 and NADPH oxidase 4 in human oral fibroblasts and by the expression of interleukin 11 in pig periosteal fibroblasts. Interleukin 11 expression was confirmed at the protein level by immunoassay. In support of TGF‐β receptor type I kinase signaling, ABL activated the nuclear accumulation of Smad‐3. The effect was independent of ABL source, mandible, calvaria or tibia. Gene expression was, however, dependent on the concentration of hydrochloric acid. ABL prepared by 0.1 M HCL was most effective compared to lysates obtained with 0.01 M and also 1.0 M HCl. Furthermore, ABL inhibited expression of osteogenic but increased chondrogenic genes. Conclusions and Clinical Implications: In summary, these results demonstrate that TGF‐β is a major growth factor being released upon preparation of demineralized bone matrix. [ABSTRACT FROM AUTHOR]

:Background: Abrasive particles in toothpastes can interact with oral cells, they can even enter periodontal pockets or extraction sockets in the oral cavity. Aim/Hypothesis: Aim of this in vitro study was to investigate whether abrasives in toothpastes can cause cytotoxicity and provoke an inflammatory cell response. Material and Methods: Abrasives of commercially available toothpastes with defined RDA values were prepared. Abrasives were obtained from toothpastes with different indication such as for kids, or highly abrasive toothpastes. L‐929, and RAW264.7 cells were exposed to various concentrations of abrasives. Cell viability testing, evaluation of gene expression, and immunoassay were performed. Results: We report here that all abrasives reduced cell viability at 0.2%. Abrasives caused a potent inflammatory response indicated by the increased expression of IL1a, IL1b and IL6 in L929 and RAW264.7 cells. In L929 cells IL1a, IL1b and IL6 expression was highest for the abrasives with the lowest and medium relative dentin abrasion (RDA) values. In RAW264.7 cells the inflammatory response of the respective genes was highest for the abrasives with medium RDA values. On protein level, the increased expression of IL6 was confirmed. Conclusions and Clinical Implications: Together these findings suggest that under in vitro conditions abrasives from toothpastes reduce cell viability and provoke an inflammatory response. [ABSTRACT FROM AUTHOR]